Immobilized hemoglobin, and processes for extracting oxygen from fluids using the same

ABSTRACT

An oxygen carrier, capable of reversibly binding and releasing oxygen, immobilized in a polymer matrix and a method of recovering dissolved oxygen from fluids utilizing the same.

This invention was developed under a contract from the Office of NavalResearch.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a material for and a process of extractingoxygen from fluids, e.g., gases and natural waters, such as, in whichthe oxygen is dissolved.

2. Description of the Prior Art

One of the primary problems which hinders man in his efforts to exploreand develop the ocean realms is the lack of a ready supply of oxygen. Inmost of the world's oceans, the oxygen content of both shallow and deepwaters is similar to that of surface water in equilibrium with air.Practical methods have not yet been devised for extracting and utilizingthis vast amount of oxygen for the maintenance of man in an underseaenvironment. Fish, however, have obviously solved the problem of oxygenextraction from seawater. Fish species weighing well over a thousandpounds and burning metabolities at rates roughly comparable to that ofman easily extract adequate dissolved oxygen from seawater for theirvaried activities. Moreover, many species of fish transfer oxygen fromseawater into a gaseous state. These fish, ones that possess swimbladders, are able to pump and concentrate oxygen against enormoushydrostatic pressure gradients. In certain fish species oxygen istransported from the dissolved state in seawater, with a _(p) O₂ of 0.2atmospheres, to a gaseous phase in the swim bladder where the _(p) O₂may exceed 100 atmospheres. The transfer of oxygen from the seawater tothe swim bladder is made possible by the presence of specializedhemoglobin molecules in fish erythrocytes. These specialized hemoglobinmolecules-called Root effect hemoglobins- act as miniature molecularpumps. The driving force for such a pump is metabolically producedlactic acid and various organic phosphate cofactors. However, we cannotdirectly mimic these biological systems, since the hemoglobin iscirculated in the blood and is consequently not in a form which can beeasily manipulated in large scale flow systems. Many attempts to developmethodologies of extracting oxygen from gaseous mixtures or water areknown. Warne et al, U.S. Pat. No. 2,217,850, and Fogler et al, U.S. Pat.No. 2,450,276, disclose processes of separating oxygen from other gasesusing solutions of cobalt compounds. However, these techniques would beineffective in a liquid system, e.g., seawater, since the compounds arein solution and would be washed away. Miller, U.S. Pat. No. 3,230,045,discloses using oxygen-binding chromoproteins such as hemoglobin andhemocyanin to separate oxygen from other gases. The chromoproteins arekept moist or in solution and are immobilized on filter paper where theymay be bound by a binder such as fibrin, and an electrolyte such assodium chloride may be present. However, this technique would also beineffective in a liquid system since the protein is not insoluble andthus would be washed away if water was allowed to flow through thesystem. Moreover, there is no provision for regeneration of oxidized(inactive) oxygen carriers. Bodell, U.S. Pat. No. 3,333,583, and Robb,U.S. Pat. No. 3,369,343, disclose apparatus for extracting oxygen fromseawater using thin tubes of silicone rubber or membrane of siliconerubber, respectively. However, neither the capillary networks nor thepermeable membranes have been found to be practicable in real-lifesituations. Isomura, U.S. Pat. No. 3,377,777, discloses concentratingoxygen from natural waters by equilibration with exhaled gases, i.e. byutilizing large areas of gas-water interface and simple diffusionalconsiderations such that the partial pressure of the gas phase and thepartial pressure of the liquid phase in the extraction zone provide forrelease of oxygen from the liquid phase into the gas phase andabsorption of CO₂ by the water phase. Additionally, the solubility ofoxygen in seawater is decreased by heating the seawater and this heatingalso increases the solubility of CO₂. However, the heating of theseawater produces an energetically undesirable process. Rind, U.S. Pat.No. 4,020,833, discloses an oxygen source for closed environmentscomprising a mixture of a metallic superoxide, which releases oxygenupon contact with CO₂ and water vapor, and a material which absorbs CO₂.However, this system suffers from the defect of the capacity beinglimited by the bulk amount of mixture which can be carried. Iles et al,U.S. Pat. No. 4,165,972, discloses separating oxygen from gas mixturesusing metal chelates as sorbents. However, the technique is notextendable to the extraction of oxygen from water.

Many compounds in solution have been examined with respect to theiroxygen absorption properties and the mechanistics thereof. Theproperties of hemoglobins, hemerythrins and hemocyanins, the naturallyoccurring oxygen carriers, have been the subject of numerous studies, asdocumented in Bonaventura et al, J. Am. Zool., 20, 7 [1980] and 20, 131(1980). Artificial oxygen carriers and their properties in solution aredescribed by a number of researchers. Traylor et al, "Solvent Effects onReversible Formation and Oxidative Stability of Heme-Oxygen Complexes",J.A.C.S., 96, 5597 (1974) discloses the effect of solvent polarity onoxygenation of several heme-base complexes prepared by reduction withsodium dithionite or a mixture of Pd black and calcium hydride.Crumbliss et al, "Monomeric Cobalt-Oxygen Complexes", Science, 6, June1969, Volume 164, pp. 1168-1170, discloses Schiff base complexes ofCo(II) which form stable cobalt-oxygen species in solution instead ofcobalt-oxygen-cobalt bridged complexes. Crumbliss et al, "MonomericOxygen Adducts of N,N'-Ethylenebis (acetylacetoniminato)ligand-cobalt(II). Preparation and Properties " J.A.C.S. 92, 55 (1970),discloses a series of monomeric molecular oxygen carriers based oncobalt ligand complexes. Dufour et al, "Reaction of Indoles withMolecular Oxygen Catalyzed by Metalloporphyrins", Journal of MolecularCatalysis (In Press), discloses the catalysis of the oxygenation ofsimple, alkyl-substituted indoles by Co(II), Co(III), and Mn(III)meso-tetraphenyl-porphins wherein a ternary complex O₂ -CoTPP-indole isformed initially. Brault et al, "Ferrous Porphyrins in Organic Solvents.I. Preparation and Coordinating Properties", Biochemistry, 13, 4591(1974), discloses the preparation and properties of ferrousdeutereporphyrin dimethyl ester and ferrous meso-tetraphenylporphin invarious organic solvents. Chang et al, "Kinetics of ReversibleOxygenation of Pyrroheme-N-[3-(1-imidazolyl)propyl] amide", disclosesstudies on the oxygenation of pyrroheme-N-[3-(1-imidazolyl)propyl]amide, i.e. a synthesized section of the myoglobin active site. Castro,"Hexa and Pentacoordinate Iron Poryhyrins", Bioinorganic Chemistry, 4,45-65 (1974), discloses the direct synthesis of hexa and pentacoordinateiron porphyrins, i.e. the prosthetic groups for the active sites ofcertain cytochrome and globin heme proteins. Chang et al, "SolutionBehavior of a Synthetic Myoglobin Active Site", J.A.C.S., 95, 5810(1973), discloses studies on a synthesized section of the myoglobinactive site and indicates that the oxygen binding reaction does notrequire the protein. Naturally occurring oxygen carriers have beenchemically cross-linked and their properties described. Bonsen et al,U.S. Pat. No. 4,053,590, discloses a polymerized, cross-linked,stromal-free, hemoglobin proposed to be useful as a blood substitute.Morris et al, U.S. Pat. No. 4,061,736, discloses intramolecularlycross-linked, stromal-free hemoglobin. Wong, U.S. Pat. No. 4,064,118,discloses a blood substitute or extender prepared by coupling hemoglobinwith a polysaccharide material. Mazur, U.S. Pat. No. 3,925,344,discloses a plasma protein substitute, i.e. an intramolecular,cross-linked hemoglobin composition. However cross-linked hemoglobinproduces macromolecular complexes that retain many of hemoglobin'snative properties. The cross-linking of hemoglobin results in a productthat is a solution or a dispersion, is not manipulable or, in fact,insolubilized. Large scale flow-thru systems where volumes of water mustflow by or through an oxygen extracting medium cannot use hemoglobinwhich has been crosslinked because the hemoglobin is not trulyinsoluble. In other words, crosslinking does not accomplish a usefulinsolubilization, in that, even after crosslinking, the protein in itsfinal form has the characteristics of a fluid.

Numerous papers have been published on immobilization of hemoglobin andits functional consequences, but not in connection with processes forefficient oxygen extraction from fluids. Vejux et al, "PhotoacousticSpectrometry of Macroporous Hemoglobin Particles", J. Opt. Goc. Am., 70,560-562 (1980), discloses glutaraldehyde cross-linked hemoglobin and itsfunctional properties. The preparation is described as being made up ofmacroporous particles. Hallaway et al, "Changes in Conformation andFunction of Hemoglobin and Myoglobin Induced by Adsorption to Silica",BBRC, 86, 689-696 (1979), discloses that hemoglobin adsorbed on silicais somewhat different from hemoglobin in solution. The adsorbed form isnot suitable for O₂ extraction from liquids. Antonini et al,"Immobilized Hemoproteins", Methods of Enzymology, 44, 538-546 (1976),discloses standard immobilization techniques as applied to hemoglobinand their functional consequences. Mention is made of hemoproteins boundto cross-linked insoluble polysaccharides such as Sephadex or Sepharose,using a pre-activation of the resin with CNBr. Rossi-Fanelli et al,"Properties of Human Hemoglobin Immobilized on Sepharose 4B", Eur. J.Biochemistry, 92, 253-259 (1978), discloses that the ability of thehemoglobin to be bound to Sepharose 4B is dependent upon theconformational state of the protein. Colosimo et al, "TheEthylisocyanate (EIC) Equilibrium of Matrix-Bound Hemoglobin", BBA, 328,74-80 (1973), discloses Sephadex G-100, Sephadex DEAE-A50 and SephadexCM-C50 as supports for human hemoglobin insolubilization. The papershows that the affinity of the insolubilized protein for EIC isincreased relative to that in solution. Lampe et al, "Die Bindung vonSauerstoff an tragerfixiertes Hamoglobin", Acta Biol. Med. Germ., 33,K49-K54 (1974), discloses studies on CM-Sephadex insolubilizedhemoglobins. Lampe et al, "Der EinfluB der Immobilisierung vonHamoglobin auf dessen Sauerstoffindung", Acta Biol. Med. Germ., 34,359-363 (1975), discloses studies on CM-Sephadex insolubilizedhemoglobins. Pommerening et al, "Studies on the Characterization ofMatrix-Bound Solubilized Human Hemoglobin", Internationales Symposiumuber Struktur und Funktion der Erythrozyten (Rapoport and Jung, ed.),Berlin Akademie-Verlag Press, 179-186 (1975), disclosesSepharose-Sephadex types of insolubilization. Brunori et al, "Propertiesof Trout Hemoglobin Covalently Bound to a Solid Matrix", BBA, 494(2),426-432, discloses Sepharose 4B or Sephadex G-200, activated by CNBr, toimmobilize the hemoglobin. Some changes in the functional properties ofthe hemoglobin were found.

As may be discerned, there are generally two classes of "insolubilized"hemoglobins described in patents or in open literature. First,cross-linked hemoglobin, e.g., as by glutaraldehyde. Biodegradation ofsuch forms of insolubilized hemoglobin would be rapidly accomplished bythe microorganisms in seawater. Nor has full functionality beendemonstrated in published accounts. This does not mean that functionalproperties are necessarily eliminated, but, that methods as describedare not suitable for achieving an immobilized form with unimpairedfunction. Second, Sephadex or Sepharose bound hemoglobins. Lowhemoglobin content per volume (specific capacity) makes these methods ofinsolubilization untenable for large scale use. Biodegradation problemsare also present. Additionally, it is not generally possible to achievehigh flow rates through such materials.

Various techniques for the insolubilization (or immobilization) ofbiological materials have been developed, though not described inconjunction with insolubilization and utilization of oxygen carriers.Stanley, U.S. Pat. No. 3,672,955, discloses a technique for thepreparation of an insoluble, active enzyme, a biological catalyst,wherein an aqueous dispersion of the enzyme is emulsified with anorganic polyisocyanate, mixed with a solid carrier and the volatilecomponents are then evaporated from the mixture. Wood et al, U.S. Pat.No. 3,928,138, discloses a method of preparing a bound enzyme wherein,prior to foaming, an isocyanate-capped polyurethane is contacted with anaqueous dispersion of enzyme under foam-forming conditions, wherebypolyurethane foams containing integrally bound enzyme are obtained.Unsworth et al, U.S. Pat. No. 3,928,230, discloses the encapsulation offluids and solids by dissolving a water-insoluble polymerizable epoxymonomer in a solvent having high affinity for water; dispersing themonomer solution in water; dispersing in the so-formed aqueousdispersion the substance to be encapsulated; adding a polymerizing agentin a solvent having a higher affinity for water than for thepolymerizing agent; and polymerizing until polymerization of the monomeris complete. Wood et al, U.S. Pat. No. 3,929,574, discloses an enzymeintegrally bound to a foamed polyurethane parepared by, prior tofoaming, contacting an isocyanate-capped polyurethane with an aqueousdispersion of enzyme under foam-forming conditions, whereby polyurethanefoams containing integrally bound enzyme are obtained. Hartdegen et al,U.S. Pat. No. 4,094,744, discloses water-dispersibleprotein/polyurethane reaction products formed by admixing awater-dispersible, biologically-active protein and an isocyanate-cappedliquid polyurethane prepolymer having a linear polyester backbone underessentially anhydrous conditions to form a solution, said protein andprepolymer reacting to form a water-soluble reaction product wherein theprotein and prepolymer are bound together. Hartdegen et al, U.S. Pat.No. 4,098,645, discloses enzymes immobilized by the process of mixingthe protein and an isocyanate-capped liquid polyurethane prepolymer inthe absence of water; foaming the mixture by reacting it with water toform a polyurethane foam. Huper et al, U.S. Pat. No. 4,044,196,discloses proteins insolubilized using polymers containing maleicanhydride or di- and poly-methacrylates. Huper et al, U.S. Pat. No.3,871,964, discloses proteins insolubilized using polymers containinganhydride, di-methacrylate and a hydrophilic monomer. However, there isno disclosure in the art of an effective way to insolubilize hemoglobinor other oxygen carriers at high concentrations so as to render themactive, insoluble and manipulable.

A need therefor continues to exist for not only improved methods forinsolubilizing hemoglobin or other oxygen carrying compounds but alsofor a method of extracting the available dissolved oxygen from naturalwaters and other fluids. Such methods as will be described will also beuseful for preparing blood substitutes which are capable of reversibleoxygen binding under physiological conditions.

SUMMARY OF THE INVENTION

Accordingly, one object of the invention is to provide an insolubilizedoxygen carrier which is effective for the extraction of oxygen fromfluids, e.g., gases and natural waters, such as seawater.

A further object of the invention is to provide an oxygen carrier in aform such that oxygen can be carried from regions of high concentration,such as the lungs, and unloaded in regions of low concentration, such asthe respiring tissues.

Briefly, these objects and other objects of the invention as hereinafterwill become more readily apparent can be attained by providing oxygencarriers which have been insolubilized at high concentration by beingentrapped and/or covalently linked to a polyurethane matrix or tocomparable supports in states that are capable of reversible oxygenbinding and are regenerable in the event of oxidation.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a 40× magnification showing the nature of a polyurethanereticulated matrix in which hemoglobin is incorporated.

FIG. 2 is a 2400× magnification of the thin walls of the matrix of FIG.1 at a region of contact between two of the oval compartments.

FIG. 3 is a 4000× magnification showing the rippled surface of the thinwalls of the matrix of FIG. 1. The hemoglobin material is held withinthe walls.

FIG. 4 is a schematic diagram of an oxygen extraction process.

FIG. 5 is a schematic diagram of a laboratory oxygen recovery apparatus.

FIG. 6 is a representative oxygen loading curve.

FIG. 7 is a representative oxygen unloading curve.

FIG. 8 is a representative absorption curve for a two column system.

FIG. 9 is a schematic diagram of an absorption system utilizing a staticoxygen carrier, denoted as Hemosponge.

FIG. 10 is a schematic diagram of an absorption system utilizing apiston system capable of driving the fluid flow from the various inletsand compressing the static oxygen carrier, denoted as Hemosponge.

FIG. 11 is a schematic diagram of an absorption system utilizing a beltsystem wherein the oxygen carrier, denoted as Hemosponge, is not staticbut is transported from regions of high oxygen concentration to regionsof low concentration or to a region where oxygen release is initiated bya chemical means.

FIG. 12 depicts reversible oxygen binding by human hemoglobin inpolyurethane gel particles of sizes comparable to those of red bloodcells. Y represents fraction saturation of the oxygen carrier.

FIG. 13 shows a spectrophotometric demonstration of reversible oxygenbinding by a film of hemoglobin insolubilized in a polyurethane gel.Oxygen extraction in this case is from air. Y represents fractionalsaturation of the oxygen carrier with oxygen.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention relates to the incorporation of an oxygen carrier, whichcan be a biological macromolecule, into an insolubilized form, which canbe a polymeric matrix. More particularly, the preferred embodiment ofthe invention involves a biochemical engineering technique known asmolecular entrapment. The oxygen carrier used by man and other mammals,as well as by most other vertebrates, is hemoglobin. By molecularentrapment, hemoglobin can be made insoluble and consequently moreamenable for use in a recycling and regenerable system. Optimally,entrapment is analogous to placing a cage around the biologically activematerial. This cage, or network, entraps the material but does notrender it inactive. The entrapment insolubilizes the material andrenders it amenable to manipulation. The degree to which function ismaintained varies greatly with the type of entrapment process used. Inthe preferred polyurethane matrices of this invention, the materialretains essentially full biological activity. The preferred material forhemoglobin insolubilization is a hydrophilic polyurethane. The wordpolyurethane is all-inclusive and is used for all polymers containingurethane linkages. Most polyurethane foams do not have as their startingpoint a water soluble component, and consequently are not compatiblewith most biological materials. A special kind of polyurethaneprepolymer, one which polymerizes when in contact with water, isrequired to create the material designated as the preferred embodimentof this invention. In FIGS. 1-3 and 6-11, the insolubilized oxygencarrier is referred to as Hemosponge. Polymerization andinsolubilization of the oxygen carrier is effected by adding water tothe pre-polymer. The hydrophilic urethane prepolymer which was used tocreate the Hemosponge described here was developed by W. D. Grace andCompany and is the commercially available material HYPOL. Other urethanemonomers compatible with the water soluble nature of hemoglobin can besynthesized and these other materials may be used in creation ofHemosponge. The means by which such hydrophilic foams can be synthesizedhave been published and are in the public domain. The advantages of thistechnique are numerous. Hemoglobin in such polyurethane foams or gelscan be entrapped in high concentrations, the matrix having a variabledegree of reticulation. The matrix in the formulations described hereinis durable and amenable to all sorts of mechanical manipulation. It canbe formed into virtually any configuration. It can be cut, machined,drilled, etc. Even more importantly, in the form of a sponge orsized-gel particles, it has very good diffusional and fluid-flowcharacteristics.

During the insolubilization process, using the preferred polyurethanematrix, the amount of CO₂ liberation can be varied over a very widerange. When CO₂ liberation is abundant, the resultant material is highlyreticulated and spongy as illustrated in FIGS. 1-3. High flow rates aretypical with such reticulated forms.

When formulated with a minimum of CO₂ liberation, polyurethane gels canbe formed without reticulation. For a typical insolubilization ofhemoglobin, the procedure would be:

(1) Pack red blood cells by centrifugation.

(2) Wash cells twice with physiological saline and repack.

(3) Lyse with distilled water to a concentration of hemoglobin of about125 mg/ml and adjust the pH to 6.0 with dilute HCl.

(4) Remove red cell membranes by centrifugation to save the hemoglobinsolution.

(5) Mix 20 ml of hemoglobin solution with 4 ml HYPOL.

(6) Allow to form a non-reticulated gel at room temperature (20°-25°C.).

The resultant gel material can be ground and sorted into gel particlesof defined size. Such gel particles of approximately 0.5 mm diameterhave been found to have good diffusional and flow-rate characteristics.Good oxygen loading curves as shown in FIGS. 6-7 have been obtainedusing packed gel particle columns at flow rates of about one columnvolume per minute in devices illustrated in FIG. 5. The diffusionalcharacteristics of such columns have been found to be dependent upon gelsize, and such gel size is readily optimizable. Additionally, the use ofsized-gel particles allows the use of fluidized bed absorption, whichgreatly increases the flow rate of oxygen-containing water. Furthermore,hemoglobin can be loaded into the sized-gel particles at highconcentrations. Up to 30 g hemoglobin/1000 ml of column volume can beattained without problems with flow rates or diffusional difficulties.Overall yield of oxygen from such columns gives about 50-70% of thetheoretical maximum based on the amount of insolubilized hemoglobinwhich the column contains. The pH during gel formation is quiteimportant. The optimum pH for the insolubilization process appear to beabout pH 6.

Hemoglobin and other oxygen carriers may be insolubilized at highconcentration and with reversible oxygen binding characteristics inother than polyurethane matrices. Acrylic gels may also be used in thisinvention, especially the hydrophilic acrylates. Also reactive polymerscontaining maleic anhydride as one of the constituents are highlysuitable for covalently binding hemoglobin or alternate oxygen carriers.Such polymers give high bonding yields (1 gr/gr polymer) and theresulting material exhibits reversible oxygen binding/unloading. Thesepolymers are particularly suitable in the form of macroporous beads. Twoother reactive polymers which may be used are the epoxy type, made bypolymerization of glycidyl methacrylate as a copolymer, and theglutaronic aldehyde type, from pyridine-containing polymers reacted withcyanogen bromide. Additionally, hemoglobin and other oxygen carriers canbe covalently bound to various insoluble matrices by other techniques,such as those used for enzyme immobilization, such as are described inMethods in Enzymology, Volume 44, Immobilized Enzymes, Academic Press,New York (1976). In these alternative methods of insolubilization,successful formulations must have the characteristics of durability,resistance to biodegradation, high specific density of hemoglobin on thecarrier, high flow rates with little pressure drop, and good diffusionalcharacteristics. Thus, Sephadex or Sepharose bound hemoglobin formspreviously described are inferior methods for hemoglobininsolubilization.

Hemoglobin is, of course, by far the most common oxygen carrying proteinfound in nature. Within this context, however, it is possible to use incommercial applications any of the hemoglobins which are available inlarge quantity, e.g., human, bovine, porcine and equine hemoglobins.Further, whole blood, lysed cells, stripped or unstripped hemolysatescan be used. Modified forms of hemoglobin, i.e. high or low affinityhemoglobins, as known in the art, are also useful. Hemoglobin can betreated to manipulate its affinity. Covalent or chemical modification,prior to immobilization, or treatment of the immobilized hemoglobin withcofactors that bind tightly and alter oxygen binding affinity (these areremovable by washing the polymeric matrix with appropriate buffers) canbe used. Additives, like catalase, superoxide dismutase andmethemoglobin reductase, can be added to the solutions of hemoglobinprior to insolubilization in the polymeric matrix. These agents arenormally found in red blood cells and can be useful in conferringstructural and functional stability to the insolubilized hemoglobin.Additionally, reagents such as glycerol, which are known to impartstructural stability to proteins in solution, can be usefully added tothe solution of hemoglobin prior to incorporation into the polymericmatrix or, likewise, prior to covalent attachment to other polymericsupports.

Although hemoglobin is by far the most common oxygen carrier found innature, other types of oxygen carriers are found in a number of species.In particular hemocyanin and hemerythrins are known and useable althoughthey suffer from the deficiency of being unavailable in largequantities.

The use of synthetic oxygen carriers, such as the modified hemesdescribed earlier and other like compounds known in the art, which showreversible oxygen binding, allow the attainment of high oxygen absorbingcapacity in minimum absorber volume. These compounds are particularlyuseful when covalently bound to a polymeric matrix.

While mere contact with dissolved oxygen is sufficient for oxygenloading of the oxygen carriers of this invention,, many variations arepossible in the unloading cycle. A chemical alteration which oxidizes orinactivates the oxygen carrier is able to cause release of all of thebound oxygen. For example, ferricyanide oxidation of hemoglobin to theferric state, called in the literature methemoglobin, is a chemicalmeans for unloading the absorbed oxygen. The pertinent equations are:

    Hb.sup.FeII +O.sub.2 ⃡Hb.sup.FeII O.sub.2      (1)

    Hb.sup.FeII O.sub.2 +oxidant→Hb.sup.FeIII +O.sub.2  (2)

    Hb.sup.FeIII +reductant→Hb.sup.FeII                 (3)

where Hb^(FeII) is ferrous deoxy hemoglobin, Hb^(FeII) O₂ is ferrous oxyhemoglobin and Hb^(FeIII) is ferric (met hemoglobin. In this and otherchemical methods, it is necessary to use a regeneration cycle toreactivate the oxygen carrier. With hemoglobin, dithionite can be usedto reduce the active sites and render them reactive towards oxygen onceagain. The loading, unloading and regeneration cycles are shown in FIG.4. During the unloading cycle, it is advantageous that the quantity offluid used for regeneration, e.g. Ferricyanide solution, be appreciablyless than is required for the loading process. The volume required inthe loading process is, however, fixed by the concentration of dissolvedoxygen in the fluid being processed.

Hemosponge incorporating fish hemoglobins can be used to extract oxygenfrom seawater. Its operation on the unloading cycle can be based on thepH sensitivity of specific fish hemoglobins. irreversible binding ofspecific cofactors to normal human blood can also render humanhemoglobin pH sensitive so that pH changes can lead to oxygen unloadingwith this system as well. The cycle for oxygen release and reloading inthese systems is depicted by the following equation: ##STR1##

The unloading process need not require a chemical treatment. Simplydecreasing the oxygen pressure in the environment of the insolubilizedoxygen carrier is a highly practical approach to oxygen unloading. Thisis, in fact, the basis for oxygen unloading in physiological systems.From this it is apparent that the insolubilized oxygen carriers canfunction as blood substitutes if prepared at the same size as red bloodcells. Reversible oxygen binding by particles of 10μ average diameterwhich contain insolubilized hemoglobin in polyurethane is illustrated inFIG. 12 and Example 3. Similarly, drained columns or beds containinginsolubilized oxygen carriers can be evacuated and the bound oxygenreleased by pulling a vacuum above the material and thus reducing theoxygen pressure. In laboratory tests, more than 50% of the loaded oxygenon insolubilized hemoglobin in polyurethane gels was found to be removedby this simple procedure. In the absence of a chemical unloading stepthe system requires no regeneration. The system can then be repetitivelycycled between its loaded and unloaded conditions. Occasionally,regeneration steps using bacteriocides and reducing agents can be usefulin keeping the system in useful operating condition. The possibility ofcarrying out such steps by methods of this invention is a majoradvantage over previous art relative to oxygen extraction from gases.

A practical system may be based on a procedure whereby a fully loadedgel is evacuated and the gas is released from the gel and is then pumpeddirectly to oxygen storage devices. A mist separator can be useful inthis step. The efficiency of the unloading cycle can be improved bybleeding low pressure steam through the system to further lower theoxygen pressure and simultaneously decrease the oxygen affinity of theinsolubilized oxygen carrier.

Unloading with or without a chemical unloading step can also be effectedindirectly through a semipermeable membrane. In this case, fluid of lowoxygen content or with a chemical which causes oxygen to be released iscirculated through the fully loaded bed of absorbent and then cycledthrough a tubular bundle of selectively permeable membranes. A pump onthe other side of the membrane can be utilized to drive the releasedoxygen out of the circulating fluid and across the semipermeablemembrane into an oxygen storage chamber.

This invention has two main features. First, the invention involvesbiologically active hemoglobin or other oxygen carriers in polymericmatrices, creating what is referred to as Hemosponge. Second, theinvention involves creation of a process, based on the Hemosponge, toprovide for oxygen extraction from fluids, such as water or air, forhuman respiratory needs.

A major improvement of this invention is the simplicity of the processby which hemoglobin and appropriate cofactors or blood or blood productscan be insolubilized at high concentration and maintained in an activeand readily useful state. Since polyurethane is the preferred matrixmaterial, methods for Hemosponge preparation in this form are describedin detail, as follows. Hemosponge is prepared by making an aqueoussolution or suspension of the biological material to be entrapped,mixing it with a non-ionic detergent of low toxicity, and then mixingthe aqueous phase with a prepolymer of urethane which has thecharacteristic of being water soluble. Alternatively, the protein can belyophilized and dispersed in the dry phase prior to mixing with aqueousphase. Very high final protein concentrations can be achieved in thisway. A number of parameters can be varied by the fabricator inpreparation of specific Hemosponge products. Insofar as the physicalnature of the polyurethane foam is concerned, the variable parametersand their effect have been described in large part by W. D. Grace andCo., the manufacturer of the hydrophilic prepolymer, in a technicalbrochure entitled "HYPOL Foam Polymer--What it is and what it does:". Ashas been mentioned, the HYPOL prepolymer contrasts with conventional(hydrophobic) foam preparations, where 3 to 5 parts of water are usedper 100 parts of polymer. The amount of water used with HYPOLhydrophilic foam polmer does not have to be carefully adjusted to theapproximate stoichiometric equivalent of isocyanate content. Instead, abroad range of water to prepolymer ratios may be used--from 2,000 to20,000 percent of the theoretical amount required. Preferably, 35 to 200parts of water per 100 parts of prepolymer are used, depending on thefoam characteristics desired. Both cell structure and aestheticproperties of foams produced from HYPOL prepolymer can be controlled bychanging the amount of water, type of surfactant, etc. For example:

1. Foams ranging from cosmetic softness to rigid and from conventionalopen cell structure to fully reticulated.

2. Rapidly wetting to slow, controlled wetting foams can be formed. Suchfoams absorb and retain from 10 to 30 times their weight of water.

3. Foams with densities of from 2 lbs./ft.³ to 20 lbs./ft.³ can bereadily prepared from HYPOL prepolymer. Tensile properties are generallycomparable to those of conventional polyurethane.

In addition to these features, the HYPOL Foams have been shown to havefire retardant properties vastly superior to those of conventionalfoams. Furthermore, favorable results from toxicity tests on HYPOL havebeen reported. At appropriate levels, no deaths or adverse symptoms werenoted in animal inhalation, ingestion and dermal studies. This featureis considered relevant insofar as particles of immobilized hemoglobin of5-10 microns diameter may be useful as blood substitutes as illustratedin Example 3.

Insofar as Hemosponge requires the addition of biologically activematerials to the aqueous phase prior to polymerization, a few additionalvariables are introduced relative to the process to be followed. Theseinclude (a) the nature of the biological material (or materials), (b)the concentration of the biological material in the aqueous phase, (c)the presence or absence of dispersing agents or detergents with thebiological material, which affects the dispersal of the biologicalmaterial in addition to its effect on the uniformity of the cellstructure and cell size of the polyurethane foam, and (d) the ratio ofaqueous phase to dry phase whereby the retention of the biologicalmaterial in the foam can be regulated. Variables which can be set by thefabricator include the following:

Nature of biological material and concentration per gram of monomer

Temperature of reaction

Pressure during the reaction

Presence or absence of specific detergents or dispersal of thebiological material in the water soluble monomer.

Presence or absence of stirring during the process of catalysis

Degrees of agitation during catalysis

pH of catalysis (this is subject to a limited amount of control)

Presence of single or multiple substances in the monomer prior tocatalysis

Absolute volume of reaction mixture

Ratio of catalyst to monomer solution

In order to clarify the exact nature of the Hemosponge, in its preferredembodiment, reference will be made to photographs of Hemosponge takenwith a scanning electron microscope. FIGS. 1, 2 and 3 exemplify theformation typical when there is human hemoglobin in the aqueous phase.For formation of Hemosponge the conditions and cofactors can be variedto suit particular physical needs. A formulation illustrative of typicalformulations is:

Aqueous Phase

(a) 8 ml of Hemoglobin (50 mg protein/ml of H₂ O). The concentration ofhemoglobin is not critical and can vary from 1 mg/ml to 150 mg/ml oreven higher. The hemoglobin can be from humans or nonhumans.

(b) 5 ml distilled water.

(c) 1 ml F-68 (19% by weight in aqueous solution)

F-68 is a non-ionic detergent of proven low toxicity; produced by BASFWyandotte Corporation of Wyandotte, Michigan. The presence of detergentis to achieve good reticulation. If non-reticulated foams or if gels areto be made, no detergent is needed. Non-ionic detergents, like Tween orTriton, can be substituted for the F-68. The concentration of detergentis not critical.

Dry Phase

(a) 6 g FHP-2000 Hypol Hydrophilic Prepolymer. Hypol is a foamablehydrophilic polyisocyanate manufactured by W. R. Grace and Co., ofCambridge, Mass. Similar formulations are obtained with FHP-2001, FHP3000 and FHP 3001 prepolymers. Nonfoaming Hypol pre-polymers can also beused.

The aqueous and dry phase materials are manually stirred with a glassstirring rod for about 15 seconds. Mixing is carried out under a vacuumhood. The polymerization, carried out at room temperature, is completedin about 3 minutes and the temperature during the foaming does notexceed 35° C. After the polymerization is complete, the product isrinsed with distilled water to remove excess detergent and unreactedmaterial.

FIG. 1 is a 40× magnification showing the nature of the matrix in whichthe biologically active material, human hemoglobin, in this case, isincorporated. It shows the highly vesicular nature of the sponge, itsthin walls, and the many holes which allow fluids to flow readilybetween and through compartments. FIG. 2 shows a 2400× magnification ofthe thin walls of the sponge at a region of contact between two of theoval compartments. Closer examination of the surface of the wallsreveals a rippled surface. This is shown in FIG. 3 in a photograph at4000× magnification. The biological material is held within the walls.

FIG. 5 and FIGS. 9-11 show schematic diagrams of various laboratorydevicesus wherein the oxygen loading and unloading process can becarried out by making use of insolubilized hemoglobin or other oxygencarriers. In particular, the apparatus of FIG. 5, for oxygen extractionfrom seawater, comprises a manifold wherein various solutions(air-equilibrated seawater, deaerated seawater, dithionite in seawaterand ferricyanide in seawater) can be fed to a pump. The pump feeds thesolution to a reactor containing the immobilized hemoglobin. Thisreactor can be of the packed bed or fluidized bed type. Effluent fromthe reactor is discharged through a flow cell containing an oxygenelectrode whereby the concentration of dissolved oxygen in the effluentcan be determined. The oxygen concentration can be read directly from anoxygen meter and/or recorded on a strip chart recorder. Typical oxygenloading and unloading curves for the apparatus of FIG. 5 are shown inFIGS. 6 and 7. FIG. 7 shows a plot of dissolved oxygen in the effluentas a function of the amount of air-equilibrated seawater (7 ppmdissolved oxygen content) passed through the reactor during the loadingcycle. FIG. 7 shows a plot of dissolved oxygen in the effluent as afunction of the amount of ferricynaide/seawater solution (7 ppmdissolved oxygen content) passed through the reactor during the loadingcycle.

Using the typical experiment illustrated previously, we can make somecalculations based on the use of a twostage two column model. To do thiswe can examine the time sequence of the loading of a single column.Consider FIG. 8. After a column (column A) reaches the point at whichsignificant oxygen is found in the effluent (12 minutes as shown in thediagram) second column (column B) would be added down-stream from columnA. Flow of seawater is now through both columns in sequence until columnA is saturated (20 minutes in the diagram). At this point, column A isremoved and unloaded while column B continues on line.

During the period (12 to 20 minutes) that column B has been acting as aback-up column it has picked up the amount of oxygen indicated by thelined area in the diagram. This is equivalent to the amount of oxygen inthe cross-hatched area of the diagram and so column B is loaded to theextent that it would have been if it had been a primary column for 4minutes. Thus, the effluent from column B will contain significantoxygen 8 minutes after it has become the primary column. Column B willbe saturated 16 minutes after becoming the primary column. The timesequence is then as follows:

    ______________________________________                                        B   P     P     U   B   P   P   U   B   P   P   U                                                     Column A                                                                      P U B P P U B P P U B P Column B                      ______________________________________                                    

Each block=8 minutes

P=Primary column, first in series

B=Back-up column, second in series

U=Unload and regenerate

Thus, the total amount of resin is loaded and unloaded every 32 minutes.Assuming 70% oxygen yield (based on the concentration of hemoglobin inthe gel), 1 g of hemoglobin will deliver at each unloading 0.7×1.4 mloxygen at 22° C., 760 torr=1 ml delivered or, considering the 32 minutecycle, this will deliver 0.03 ml/min. Note that the above example ofoxygen extraction utilizes sized gel particles in which human hemoglobinis used as the oxygen carrier. The apparatus for such extraction isshown in FIG. 5 and schematically in FIG. 9. Alternatively, the manifoldof FIG. 5 can switch between solutions of air, low oxygen gas or vacuumor dithionite in 0.05 M Tris buffer or 0.2 M phosphate buffer, orferricyanide solution in such buffers to accomplish oxygen extractionfrom air. The methodology as just described is therefore applicable forboth oxygen extraction from water or from gas. A spectral demonstrationthat the insolubilized oxygen carrier, hemoglobin, in this example, canextract oxygen from air was obtained by spectrophotometric methods (seeFIG. 13).

FIGS. 9-11 illustrate additional mechanical embodiments for the oxygenextraction process. Thus the oxygen carrier can be static and variousfluids can be flowed by it as shown in FIGS. 9 and 10. Otherwise, theoxygen carrier can be cycled between areas of high oxygen content, e.g.,air or nautral waters, and areas where oxygen unloading is accomplished.This is schematically depicted in FIG. 11. The method of how the oxygencarrier can be moved from regions of high oxygen to regions whereunloading is accomplished is elaborated by FIG. 12 and Example 3, wherethe carrier is shown to perform as a blood substitute.

As noted above, the extraction of oxygen from a fluid like air can alsobe effected using a device of the sort described herin. In this mode,the supported bed of the immobilized oxygen carrier is operated with airflowing through it, rather than water. The oxygen carrier will not onlyremove oxygen from the air, but, the gas, having the oxygen removed,will be greatly enriched in N₂ (see the table below)

    ______________________________________                                                   % Composition                                                                          Air (quantitative                                         Gas          Air    removal of O.sub.2)                                       ______________________________________                                        O.sub.2      21     --                                                        N.sub.2      78     98.73                                                     Ar           0.93   1.18                                                      other        0.07   0.09                                                      ______________________________________                                    

A demonstration that the immobilized carrier is capable of extractingoxygen from air is clearly obtainable by spectrophotometric analysis asshown in FIG. 13. A film of insolubilized oxygen carrier, hemoglobin, inthis case, was reversibly cycled between low and high oxygenconcentration. The saturation of the carrier with oxygen was determinedspectrophotometrically and from such measurements it is possible todetermine loading-unloading characteristics of devices made of suchfilms. In the example of FIG. 13, half saturation occurs at an oxygenpressure of 1.26 mm Hg. As noted previously, these characteristics aresubject to modification by using different oxygen carriers or bychemically modifying human hemoglobin.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLE 1

The following tables are those which represent measurements of theefficiency of an oxygen extraction device based on the piston systemdesign of FIG. 10 in which the compressibility of a reticulated matrixof Hemosponge is utilized. In the example outdated human blood was usedas the oxygen carrier in the Hemosponge and chemical recycling wascarried out as in equations I-III. As is apparent, the additivesco-incorporated with the blood in the reticulated matrix affect theefficiency of extraction.

    ______________________________________                                                                     % O.sub.2 IN GAS                                 HEMOGLOBIN ADDITIVE                                                                              Wt. Nb    RECOVERED                                        ______________________________________                                        5% GLYCEROL        3.5 GM    23.4%                                            10% GLYCEROL       3.5 GM    22.35                                            CATALASE           3.5 GM    23.5%                                            CATALASE + 2% GLYCEROL                                                                           3.5 GM    22.1%                                            CATALASE, 2% GLYCEROL,                                                        and 5 mM INOSITOL                                                             HEXAPHOSPHATE      3.5 GM    21.7%                                              "                7.0 GM    27.1%                                              "                10.0 GM   23.6%                                            ______________________________________                                    

EXAMPLE 2

This example illustrates the process and method whereby hemoglobin, anoxygen carrier, is bound to a support, that is not polyurethane, in highconcentration and with biological activity such that oxygen extractioncan be achieved.

A mixture of 7 g of tetraethylene glycol dimethacrylate, 2 g methacrylicacid, 1 g maleic anhydride, 0.1 g of azoisobutyronitrile was dissolvedin 100 ml of benzene and warmed to 60° C. for 4 hours. Thereafter, 0.1 gof azoisobutyronitrile was added and the mixture was heated at 70° C.for 2 hours and then at 80° C. for 2 hours. The polymer whichprecipitated was filtered off, washed with petroleum ether and dried ina vacuum to give 8.5 g of polymer.

One gram of the polymer prepared by this procedure was mixed with 10 mlof a hemoglobin solution, containing 120 mg hemoglobin per ml and 20 mlof water. The pH was adjusted to 6.3 and maintained at pH of 6.3-6.5 byaddition of NaOH. After 24 hours, the polymer bound hemoglobin wasremoved by filtration.

Hemoglobin in filtrate+washing: 72 mg

Hemoglobin bound to polymer: 1.13 g

The bound hemoglobin was reduced with dithionite and then oxygenated bybubbling air through a suspension thereof. It was then treated withexcess potassium ferricyanide solution:

O₂ released=0.78 mg

Theoretical amount of O₂ bound by hemoglobin in polymer=1.92 mg

Yield=41%

EXAMPLE 3

This example illustrates the process and method whereby hemoglobin, anoxygen carrier, can be insolubilized in polyurethane particles ofdimensions like those of red blood cells (5-10μ) and perform as a bloodsubstitute.

In this case, 20 ml of human hemoglobin at a concentration of 125 mg/mlwas mixed with 4.0 ml of HYPOL (Grace Co.) non-foaming prepolymer andallowed to polymerize at room temperature as a gel. The gel was groundto provide particles and then sized. Such particles in the size range ofabout 0.5 mm were used in columns for oxygen extraction from seawater asdescribed in detail in the text (FIG. 5). Smaller particles wereobtained by repetitive screening and preparative centrifugation.Particles with dimensions comparable to those of red blood cells (5-10μ)were obtained and the oxygen binding properties of such particles, in0.2 M phosphate buffer, pH 7.0, at 20° C., were examined. FIG. 12illustrates an oxygen binding curve for such particles and demonstratesthat such may be considered as blood substitutes. Thus, for theinsolubilized hemoglobin in small particles, it was found that halfsaturation was attained at an oxygen pressure of 3.7 mm Hg andcooperativity, expressed by the Hill coefficient, was 1.7. In humanhemoglobin, in solution under comparable conditions of temperature,buffer and pH, the oxygen pressure for half saturation was 2.4 mm Hg andcooperativity was expressed by a Hill coefficient of 2.5.

Having now fully described this invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionset forth herein.

We claim:
 1. An oxygen carrier, capable of reversibly binding andreleasing oxygen, immobilized in a polymer matrix selected from thegroup consisting of compounds having urethane linkage, acrylic gels,maleic anhydride containing polymers, epoxy type polymers, glutaronicaldehyde type polymers and mixtures thereof.
 2. The immobilized oxygencarrier of claim 1, wherein said oxygen carrier is hemoglobin, ahemocyanin a synthetic heme, an organo-metallic compound or a chemicallymodified hemoglobin or hemocyanin.
 3. The immobilized oxygen carrier ofclaim 1, wherein said polymer matrix is a hydrophilic acrylate gel. 4.The immobilized oxygen carrier of claim 1, wherein said polymer matrixis a polyurethane.
 5. The immobilized oxygen carrier of claim 4, whereinsaid polyurethane is a foam.
 6. The immobilized oxygen carrier of claim4, wherein said polyurethane is a gel.
 7. The immobilized oxygen carrierof claim 6, wherein said polyurethane gel is in the form of sized gelparticles.
 8. The immobilized oxygen carrier of claim 7, wherein saidsized gel particles have a diameter in the range of about 5 to 10microns.
 9. The immobilized oxygen carrier of claim 1, wherein saidoxygen carrier is hemoglobin.